Human Sigmoid Colon

8/13/2019 Human Sigmoid Colon

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Effect of otilonium bromide on contractile patterns in the

human sigmoid colon

D. GALLEGO,* M. AULI,, J. ALEU, E. MARTINEZ ,, L. ROFES,* J. MARTI-RAGUE, M. JIMENEZ *, & P. CLAVE*,,

*Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (Ciberehd), Instituto de Salud Carlos III,

Barcelona, Spain

Department of Surgery, Hospital de Mataro, Mataro, Spain

Fundacio de Gastroenterologia Dr. F. Vilardell, Barcelona, Spain

Department of Cell Biology, Physiology and Immunology, Universitat Autonoma de Barcelona, Barcelona, Spain

Abstract

BackgroundThe mechanism of action of the spas-molytic compound otilonium bromide (OB) on human

colonic motility is not understood. The aim of our

study was to characterize the pharmacological effects

of OB on contractile patterns in the human sigmoid

colon. Methods Circular sigmoid strips were studied

in organ baths. Isolated smooth muscle cells from

human sigmoid colon were examined using the cal-

cium imaging technique. Key Results Otilonium bro-

mide inhibited by 85% spontaneous non-neural

rhythmic phasic contractions (RPCs), (IC50 = 49.9 n-

mol L)1) and stretch-induced tone (IC50 = 10.7 n-

mol L)1) with maximum effects at micromolar range.

OB also inhibited by 50% both on- (IC50 = 38.0 n-

mol L)1) and off-contractions induced by electrical

stimulation of excitatory motor neurons. In contrast,

the inhibitory latency period prior to off-contractions

was unaffected by OB. OB inhibited acetylcholine-,

substance P-, and neurokinin A-induced contractions.

The L-type Ca2+ channel agonist BayK8644 reversed

the effects of OB on RPCs, on- and off-contractions.

Hexamethonium, atropine, the NK2 antagonist, or

depletion of intracellular Ca2+ stores by thapsigargin

did not prevent the inhibitory effect of OB on RPCs

and electrical contractions. KCl-induced calcium

transients in isolated smooth muscle cells were also

inhibited by OB (IC50= 0.2 lmol L)1

).Conclusions &Inferences Otilonium bromide strongly inhibited the

main patterns of human sigmoid motility in vitro by

blocking calcium influx through L-type calcium

channels on smooth muscle cells. This pharmacolog-

ical profile may mediate the clinically observed effects

of the drug in patients with irritable bowel syndrome.

Keywordsgastrointestinal motility, sigmoid colon,

smooth muscle, spasmolytic drugs.

INTRODUCTION

Irritable bowel syndrome (IBS) is a chronic functionalgastrointestinal disorder affecting up to 11.5% of the

general population1 and is characterized by abdominal

pain or discomfort associated with a change in bowel

habit. Altered motility in the small bowel and colon

may contribute to a change in bowel behavior, and a

combination of increased motility and spasm, visceral

hypersensitivity, and abnormalities in central pain

processing may explain the origin of the abdominal

pain.2 Recent in vivo studies showed that pain hyper-

sensitivity and colon hypermotility including

increased phasic motility and enhanced smooth mus-

cle tone are independent factors contributing to

symptoms in patients with IBS.3 Antispasmodics and

muscle relaxants are widely used as first-line treat-

ment in these patients. However, the biological ratio-

nale for the efficacy of antispasmodics is unclear, and

their pharmacological effects may vary among com-

pounds. Recent studies found that antispasmodics may

act by reducing colonic contraction and increasing

transit time and therefore reducing pain and stool

frequency in patients with IBS.2 Among antispasmo-

dics, otilonium bromide (OB), a quaternary ammonium

Address for correspondence

Pere Clave, MD, PhD, Associate Professor of Surgery,Department of Surgery, Hospital de Mataro, UniversitatAutonoma de Barcelona, C/Cirera s/n. 08304, Mataro, Spain.Tel: +34 93 741 77 00; fax: +34 93 741 77 33;e-mail: [email protected] study was presented in part at the 21st InternationalSymposium on Neurogastroenterology and Motility, Jeju,Korea, August 2007.Received: 15 October 2009

Accepted for publication:22 February 2010

Neurogastroenterol Motil (2010) 22, e180e191 doi: 10.1111/j.1365-2982.2010.01495.x

2010 Blackwell Publishing Ltde180


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derivative compound, has shown consistent evidence

of efficacy on IBS patients.2,4,5 Clinically, the main

effects of OB include a global improvement of IBS

symptoms, a reduction in the frequency and severity of

abdominal pain, and an increase in the distension pain

threshold with the use of a sigmoid balloon.69

How-ever, the effects and the mechanism of action of OB on

human colonic motility are not fully understood, and

the few studies that have investigated the mechanisms

of action of OB in animal models described a hetero-

geneous pharmacological profile.5,10

Most studies on the pharmacodynamics of OB have

been conducted on rodents and suggest several mecha-

nisms of inhibition of OB on spontaneous and stimu-

lated intestinal motility. Animal studies found that OB

inhibited contractions induced by acetylcholine (ACh),

serotonin, substance P (SP), histamine, barium chloride

(BaCl2) a n d K+ and described how OB exerted its

spasmolytic action by a combination of antagonistic

actions at muscarinic receptors, by blocking L-type Ca

channels, by antagonizing tachykinin NK1 and NK2receptors and by blocking nicotinic-mediated

responses;5,1012 however this has not been fully char-

acterized in humans. We haverecently characterized the

different types of contractions in human sigmoid colon

in vitro. Circular strips of human sigmoid colonin vitro

developed spontaneous rhythmic phasic contractions

(RPCs) of non-neural origin and responded to electrical

stimulation (EFS) of enteric motor neurons (EMNs) with

twodistinctpatterns of strongcontractile responses: (i)a

phasiccontractionduring EFS on-contractions or (b)aphasic contraction after EFS off-contraction caused

by stimulation of excitatory EMNs following a period of

latency caused by stimulation of inhibitory EMNs

releasingNOandATPactingatP2Y1 receptors.13Ampli-

tude of electrical-induced contractions depended on

stimulation of excitatory EMNs releasing ACh and

tachykinins acting on muscarinic and NK2 receptors

located on smooth muscle cells.13

The aim of this study was to assess the pharmaco-

dynamics and effects of the spasmolytic agent OB on

the main types of in vitro contractions in human

sigmoid colon and to pharmacologically characterize

OBs mechanism of action.

METHODS

Tissue specimens

Tissue specimens of human sigmoid colon were obtained from 70

patients without symptoms of major clinical motility disorders

who underwent surgery for rectal cancer (T2/T3 stages, age range3285 years, 44% women). All strips were obtained from macro-

scopically non-invaded regions. The experimental protocols were

approved by the Institutional Review Board of the Hospital de

Mataro, Barcelona, Spain.

Preparation of circular muscle strips andisometric tension recording

The colonic segment was cut open longitudinally along the

mesenteric border, the mucosal layer was removed and trans-

mural muscle strips (3 mm wide by 10 mm long) were cut inthe direction of the circular muscular fibers. Weight of the

strips was 0.21 0.01 g, N = 31. A silk thread was attached to

either end of the strips and they were placed in 10 mL organ

baths filled with Krebs solution (37 C, bubbled with a mixtureof 5% CO2/95% O2, pH 7.4) as described in previous stud-

ies.13,14 Strips were positioned between two parallel platinum

wire electrodes 10 mm apart and changes in tension weremeasured using isometric force transducers, recorded on a chart

recorder (model 03 Force Transducer and model 7 Series

Polygraph; Grass Instruments Co, Quincy, MA, USA) anddigitized (Acqknowledge, MP100; Biopac Systems, Inc, Goleta,

CA, USA). In each experiment, up to six strips from the same

specimen were simultaneously studied. Strips were initiallystretched to 4 g of force and equilibrated for 1 h and then the

following patterns of colonic motility were assessed.

Spontaneous RPCs After the equilibration period, strips devel-

oped spontaneous RPCs. Origin of RPCs was characterized by

the ganglionic blocker hexamethonium 100 lmol L)1, the neu-ral blocker tetrodotoxin (TTX) 1 lmol L)1 that interrupts Na+

action potentials along the axon of EMNs, withdrawal ofextracellular Ca2+ from the medium (by using a Ca2+-free

Krebs solution with EGTA 1 mmol L)1), and depletion of

intracellular Ca2+ stores by 20-min incubation with thapsigargin

10 lmol L)1, a blocker of sarco/endoplasmic reticulum Ca2+

ATPase.15

Stretch-induced responses After the period of equilibration,stretch was applied to strips from 0 to 7 g in steps of 1 g each

30 min. Baseline tone and amplitude of RPCs was measuredduring the last 10 min of each stretch. To avoid neural mediated

responses, this study was performed in the presence of the neuralblocker TTX (1 lmol L)1).

Contractions induced by EFS of EMNsElectrical stimulation was

applied by an electrical stimulator (Model S88; Grass Instru-

ments Co) and a power booster (Stimu-Splitter II; Med-Lab

Instruments, Loveland, CO, USA).13 Ten-second trains of pulsesof 0.4 ms duration at 140 Hz and 26 V were delivered to the

electrodes and simultaneously recorded on tension tracings

through a synchronized TransistorTransistor logic signal be-tween the electrical stimulator Grass S88 and the computerized

Biopac System. In a previous study, we found sigmoid stripsresponded to EFS with two distinct patterns of contractile re-sponses of neural origin (a) a phasic contraction which began

during EFS on-contractions or (b) a phasic contraction which

began after EFS off-contractions.13 In the present study, theseEFS responses were further characterized by hexamethonium

100 lmol L)1, and depletion of extracellular and intracellular

Ca2+ stores.

Direct contractions induced by excitatory neurotransmittersIn a

previous study, we found that sigmoid contractions induced bystimulation of excitatory EMNs were mediated by released ACh

and tachykinins mainly acting on muscarinic and NK2 receptors

Volume 22, Number 6, June 2010 Otilonium bromide inhibits human sigmoid motility

2010 Blackwell Publishing Ltd e181


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located on smooth muscle cells.13 In this study, we assessed the

direct effect of ACh (0.01100 lmol L)1), SP (0.00110 lmol L)1),and neurokinin A (NKA) (1 nmol L)1 to 1 mmol L)1) on sigmoid

strips.

Calcium image technique

Smooth muscle cells were isolated by a mechanical and enzy-

matic process. Briefly, the circular muscular layer was dissected

and cut into small pieces (12 mm). The muscle pieces were

placed in Ca2+-free Hanks solution (NaCl 115 mmol L)1, KCl5.4 mmol L)1, MgCl26H2O 0.5 mmol L

)1, NaHCO34.2 mmol L)1,

Na2HPO4 0.3 mmol L)1, KH2PO4 0.4 mmol L

)1, MgSO47H2O

0.4 mmol L)1, glucose 5 mmol L)1). They were incubated with1.5 mg mL)1 papain (Sigma, St Louis, MO, USA), 1 mg mL)1

bovine serum albumin (BSA) and 2 mg mL)1 soy bean trypsin

inhibitor (SBTI) for 3 min at 32 C and stirred at 108 g.16,17 Aftercentrifugation, the muscle pieces were incubated in 1 mg mL)1

BSA, 0.75 mg mL)1 collagenase type II (Worthington, Lakewood,

NJ, USA) at 32 C and stirred again at 27 g until isolated cellsappeared. Isolated smooth muscle cells were placed in a recording

medium (NaCl 145 mmol L)1, KCl 4.8 mmol L)1, MgCl26H2O 1

mmol L)1, CaCl2 1.8 mmol L)1, glucose 10 mmol L)1, HEPES

10 mmol L)1, pH 7.4). Fluo-4/AM (Teflabs, Inc., Austin, TX, USA)was used to monitor changes in the calcium level in cytosol. Fluo-

4 (50 lg) was dissolved in DMSO (25 lL) and 2 lL of this stock

solution was added to each culture plate in 1 mL recording

medium for 45 min. After washing out the remaining dye, cellswere incubated in the recording medium. The cells were imaged

with IX-FLA equipment (Olympus Biosystems, Heidelberg,

Germany) connected to an Olympus IX70 microscope with an20 lens. The cells were scanned using CellR software (Olympus

Biosystems). Cells were exposed briefly (5 s) to high potassium

solution (75 mmol L)1 KCl) causing a sudden increase in transient

intracellular calcium [Ca2 + ]i in the smooth muscle cells(control), and the effect of 10 min incubation of cumulative

concentrations of OB (10 nmol L)1 to 100 lmol L)1) was assessed.

Experimental design

We assessed the effect of OB (10 pmol L)1 to 100 lmol L)1) on the

main human sigmoid motility patterns: (i) spontaneous RPCs; (ii)

stretch-induced tone; (iii) contractions induced by EFS of EMNs

EFS on and off contractions ; (iv) direct contractions inducedby ACh, SP and NKA, and (v) calcium transients induced by KCl

in isolated muscle cells. We compared the effects of OB with those

caused with the specific L-type channel antagonist nifedipine(10 pmol L)1 to 10 lmol L)1), atropine (1 pmol L)1 to 1 lmol L)1),

and the NK2 receptor antagonist GR 94800 (1 pmol L)1 to

1 lmol L)1). We also characterized the mechanism of action ofOB by pharmacological studies using the specific L-type channel

agonist BayK8644 (1 nmol L)1 to 10 lmol L)1), and by assessing

the effect of OB on RPCs and EFS contractions following blockadeof nAChRs by hexamethonium 100 lmol L)1, blockade of mus-

carinic receptors by atropine 1 lmol L)1, blockade of NK2 recep-

tors by the NK2 receptor antagonist GR 94800 (1 lmol L)1) and

following depletion of intracellular calcium stores by thapsigargin10 lmol L)1.

Drugs

Otilonium bromide was obtained from Laboratorios Menarini SA,

(Badalona, Spain). Acetylcholine chloride (ACh), SP, BayK8644,nifedipine, hexamethonium, BSA, SBTI from Sigma; TTX,

atropine sulphate, from Research Biochemicals International

(Natick, MA, USA); NKA, thapsigargin, NK2 receptor antagonistGR 94800 from Tocris, (Bristol, UK); Fluo-4 AM from Teflabs Inc.

Thapsigargin, n-butyl-hyoscine 20 mg per 1 mL from BoehringerIngelhemim Espana SA, (Barcelona, Spain). Stock solutions were

made by dissolving drugs in distilled water except for thapsigargin

and Fluo-4 AM which were dissolved in DMSO (0.01%) and

BayK8644 which was dissolved in ethanol (0.01%).

Data analysis and statistical procedures

Spontaneous RPCs were measured as the area under the curve

(AUC), in g min)1 and paired Students t-test was used to assess

the effect of antagonists and agonists on the proposed putativeneurotransmitters. The effect of antagonists on RPCs was

assessed following 20 min incubation, the last 10-min period

being analyzed. Data were normalized with respect to a controlperiod (10 min before addition of the antagonist) and concen-

tration response curves analyzed by two-way repeated measureANOVA analysis. The effect of agonists was measured as theAUC of the 2-min period following their addition to the bath.

The concentrationresponse curve for each agonist was com-

puter fitted using nonlinear regression, and the IC50 was

calculated (GRAPHPAD PRISM, version 4.01; GraphPad Prism Soft-ware, San Diego, CA, USA). Latency of EFS contractions was

defined as the period of time from the beginning of EFS to the

onset of contraction; and maximal amplitude of EFS contrac-

tions (in g) was also measured. The effect of OB and the otherdrugs on latency and amplitude of on- and off-contractions was

analyzed by two-way ANOVA for repeated measures. When the

two-way ANOVA was significant, the Bonferroni test was carriedout to determine the frequencies or doses of statistically

different responses. Data are expressed as mean SEM. Changes

in the Fluo4 fluorescence were recorded for 30 s at 2.5 Hz

with a spatial resolution of 512 480 pixels. At the end ofthe experiments, the images were analyzed over time by

CellR software (Olympus Optical Co., Ltd, Tokyo, Japan) using

regions of interest (ROIs). Fluorescence intensity was normal-ized to the basal fluorescence at the onset of the recording for

each ROI, and peaks were analyzed as previously described.

Paired Students t-test or ANOVA test was used before and afterdrug addition. A P < 0.05 was considered statistically signifi-

cant. N values indicate the number of samples from different

patients.

RESULTS

Effects of OB on spontaneous RPCs

Human circular sigmoid strips developed spontaneous

RPCs after 1 h equilibration. The AUC, amplitude, and

frequency of RPCs were 72.85 10.87 g min)

1, 3.53 0.52 g, and 2.48 0.24 contractions min)1 (N= 20)

respectively. The activity of RPCs(AUC) was unaffected

by 30-min exposure to the neural blocker TTX

1 lmol L)1 (+6.16 1.5%. ns, N = 11) or hexametho-

nium (100 lmol L)1) ()3.1 23.57%, ns,N= 5). Block-

ade of muscarinic receptors by atropine (1 pmol L)1 to

1 lmol L)1) or hyoscine (4.5 pmol L)1 to 4.5 lmol L)1)

and blockade of NK2 receptors by the NK2 receptor

antagonist GR 94800 (1 pmol L)11 lmol L)1) caused a

D. Gallegoet al. Neurogastroenterology and Motility



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significant reduction of RPCs, an effect dependent on

theirconcentration,the design of the experiment (cumu-

lative vs single concentrations)and the timeof exposure,

but did not block them (Fig. 1). All these results suggest

that RPCs have a non-neural originbut aremodulated by

EMNs. RPCs were greatly reduced ()

62.54 18.89%,P < 0.01;N= 6), following 30 s removal of extracellular

calcium and in contrast were only slightly reduced by

depletion of intracellular calcium stores by 30-min

incubation with thapsigargin 10 lmol L)1 ()26.47

16.76%, ns; N= 5) indicating the importance of extra-

cellular calcium in the origin of this pattern of contrac-

tions. RPCs were almost abolished by OB ()84.17

16.87% at 100 lmol L)1) in a concentration-dependent

manner (IC50of 49.9 nmol L)1,N= 8). RPCs were also

abolished ()96 1.75% at 10 lmol L)1) by the L-type

calcium channel blocker, nifedipine, with an IC50 of

1.00 nmol L)1 (N= 7) (Fig. 1, Table 1).

The specific L-type channel agonist, BayK8644

(1 nmol L)1 to 10 lmol L)1), increased the amplitude

of RPCs in a concentration-dependent manner

(1 nmol L)1 to 1 lmol L)1, EC50= 0.07 lmol L)1); and

at10 lmol L)1 this excitatory effect was reduced (N= 3)

(Fig. 2). BayK concentration dependently reversed the

inhibitory effects of nifedipine and OB (10 lmol L)1) on

RPCs (Fig. 2). In addition, OB 100 lmol L)1 strongly

inhibited RPCs following blockade of muscarinic recep-

tors by atropine 1 lmol L)1 ()64.12% 11.62, P < 0.05,

N= 5), blockade of NK2receptors by the antagonist GR

94800 ()70.49 10.32%, P < 0.0001, N= 7), depletion

of intracellular calcium by thapsigargin 10 lmol L)1

()75.16 7.12%P < 0.05,N= 5), and blockade of nico-

tinic receptors by hexamethonium 100 lmol L)1

()86.49 9.23%P < 0.05,N= 4) (Fig. 3).

Effect of OB on stretch-induced contractions

Stretch of strips did not modify the frequency of sponta-

neous RPCs. On the contrary, basal tone of strips and

amplitude of RPCs were progressively increased by

stretch (N= 15, ANOVAP < 0.001). OB was tested at six

differentconcentrations from1 nmol L)1 to 1 lmol L)1,

N = 5. Amplitude of stretch-induced contractions was

decreasedby OB, significant differences wereobservedat

the concentration of 1 lmol L)1 ()64.21%). (From

36.08 7.5% in control conditions to 12.9 8.1 after

incubationwithOB1 lmol L)1,at7 gofinducedstretch,

N= 5 P < 0.001.) Stretch-induced tone was also reduced

by OB, with significant differences observed at 0.1

lmol L)1 ()52.76%) and 1 lmol L)1 ()55.37%; Fig. 4).

(From3.07 0.23 gincontrolconditionsto1.45 0.18 g

with OB 0.1 lmol L)1,N= 5 and to 1.37 0.29 g with

OB 1 lmol L)1, N= 5, P < 0.001 each.)

Effect of OB on sigmoid contractions induced byelectrical stimulation of EMNs

In a recent study, we found EFS produced two distinct

patterns of contractile responses in the human sigmoid

colon: (i) contractions which began during EFS, definedas on-contractions caused by selective stimulation of

excitatory EMNs and (ii) contractions which began after

EFS, defined asoff-contractions also caused by stimula-

tion of excitatory EMNs following a period of latency

caused by stimulation of inhibitory EMNs.13 In the

present study, OB induced a significant concentration-

dependent inhibition in the amplitude of EFS-induced

on-contractions (IC50= 0.38 nmol L)1, )49.39

13.75% at 40 HzP < 0.05,N= 6) (Table 1). Incubation

with the L-type calcium channel blocker nifedipine

abolished on-contractions (IC50= 0.13 nmol L)1,

)96.28 3.71% at 40 Hz P < 0.05, N= 6, Fig. 5). EFS

on-contractions were fully abolished following incuba-

tion with free calcium Krebs solution for 30 s (N= 6),

and were reduced by atropine (1 lmol L)1, 40 Hz,

)69.58 3.49%, N= 9, P < 0.001), n-butyl-hyoscine

100 lmol L)1 40 Hz, )49.81 8.49%,N= 6,P < 0.05),

GR94800 (1 lmol L)1) (40 Hz: )26.07 6.71% N= 6,

P < 0.001), and unaffected by hexamethonium

(100 lmol L)1) (40 Hz, )9.69 7.82%, N= 5, ns) and

thapsigargin (10 lmol L)1, 40 Hz, )10.35 14.56%,

N= 5, ns). OB 100 lmol L)1 significantly inhibited the

amplitude of EFSon-contractions following blockade of

muscarinic receptors by atropine 1 lmol L)1 ()40.47

9.28%, N= 9, P < 0.01), blockade of NK2 receptors byGR4800 1 lmol L)1 ()58.61 9.08%) blockade of nico-

tinic receptor by hexamethonium 100 lmol L)1

()47.33 9.04%) and depletion of intracellular calcium

stores by thapsigargin 10 lmol L)1 ()47.19 9.92%).

OB also induced a concentration-dependent inhibition

in the amplitude of electrical off-contractions at high

frequencies of stimulation (2040 Hz) ()58.69 15.61%

at 40 Hz, P < 0.05, N = 6). In contrast, latency of off-

contractions was unaffected by OB ()5.18 2.26%, at

40 Hz, ns, N = 6). Nifedipine 1 lmol L)1 strongly re-

duced off-contractions at 140 Hz ()67.86 12.4%, at

40 Hz, P 0.001, N= 5), an effectthat wasfully reversed

by BayK8644 (1 lmol L)1). The inhibition of the ampli-

tude of EFS on- and off-contractions caused by OB

10 lmol L)1 was also fully reversed by the calcium

channel activator BayK8644 (1 lmol L)1) (Fig. 6).

Effect of OB on the responses induced by directstimulation of sigmoid smooth muscle cells

Otilonium bromide 100 lmol L)1 significantly inhibi-

ted the direct contraction produced by ACh




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(10 lmol L)1, )74.63 8.69%; P < 0.001, N= 10) and

SP (10 lmol L)1, )78.8 6.66%; P < 0.05, N= 7) in this

preparation. Nifedipine 100 lmol L)1 also inhibited the

direct contractions induced by ACh, and SP. Contrac-

tion induced by 10 lmol L)1 NKA was also significantly

reduced by 100lmol L)1OB)61.61 4.12%; P < 0.001,

N= 4) and almost abolished by 100 lmol L)1 nifedipine

further suggesting L-type Ca2+ channels contribute to

cholinergic and tachykinergic responses. During exper-

iments with calcium image technique, application of

high extracellular potassium (75 mmol L)1) induced an

intracellular calcium increase in smooth muscle cells.

This increase was concentration dependently reduced

by OB (0.01100 lmol L)1). OB inhibited the calcium

transients with an IC50of 0.2 lmol L)1, 15 cells,N= 5)

(Fig. 7).

Figure 1 Mechanical recordings showing inhibition of spontaneous motility (RPCs) by increasing concentrations of otilonium bromide (A),nifedipine (B), atropine (C), n-butyl-hyoscine (D) and the NK2 antagonist GR94800 (E). Cumulative concentrationresponse curves of (F) L-type

calcium channel blockers, otilonium bromide and nifedipine, (G) muscarinic receptor blockers, atropine and n-butyl-hyoscine and (H) the NK2

antagonist GR94800.




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DISCUSSION AND CONCLUSIONS

In the present study, we have assessed the strong

inhibitory effect OB has on the main patterns of human

sigmoid motility in vitro (RPCs, smooth muscle tone,

contractions induced by stimulation of excitatoryEMNs, contractions induced by direct effect of excit-

atory neurotransmitters and calcium transients in

isolated colonic smooth muscle cells). In contrast, OB

did not affect inhibitory neuromuscular transmission.

OB inhibited calcium transients induced by KCl in

isolated sigmoid smooth muscle cells in a similar way

to the inhibition of inward currents driven by L-type

calcium channels in rat colonic smooth muscle cells17

and human small intestine.18 Moreover, binding stud-

A

B

C

Figure 2 (A) Mechanical recording and histograms showing the increase in the activity of spontaneous rhythmic phasic contractions (RPCs) induced

by the L-type Ca2+ channel agonist BayK8644. Incubation of strips with (B) the L-type Ca2+ channel antagonist nifedipine (10 lmol L)1) or (C)

otilonium bromide (10 lmol L)1) concentration-dependently antagonized and prevented the effect of BayK8644. AUC, area under the curve of RPCs.

Data are expressed as mean SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

Table 1 Pharmacodynamics of otilonium bromide and nifedipine on

in vitro motor patterns in the human sigmoid colon

Pattern

IC50+ SEM

(mol L)1)

Emax(% inhibition)

Otilonium

bromide

RPCs 4.99 10)8 0.14 84.17 16.87

Stretch-induced

tone (7 g)

1.07 10)8 0.69 54.92 8.21

Stretch-induced

amplitude (7 g)

7.76 10)6 0.25 64.21 4.03

EFS-on 3.80 10)8 0.20 49.39 13.75ACh contraction 2.52 10)2 0.58 74.63 8.69

SP contraction 3.16 10)4 0.45 78.8 6.66

Nifedipine RPCs 1.00 10)8 0.69 95.35 6.37

EFS-on 1.32 10)10 0.45 96.28 3.71

ACh contraction 6.92 10)8 0.31 73.56 12.51

SP contraction 5.13 10)9 0.25 89.59 7.15




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A

B

C

D

Figure 3 Representative tracings and graph bars showing how single concentrations of OB (100 lmol L)1) strongly inhibit rhythmic phasic con-

tractions following: (A) blockade of muscarinic receptors by atropine 1 lmol L)1, (B) depletion of intracellular Ca by thapsigargin 10 lmol L)1, (C)

blockade of nicotinic receptors by hexamethonium 100 lmol L)1, and (D) blockade of NK2 receptors by GR 94800. Data are expressed as mean SEM

(*P < 0.05, **P < 0.01).




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ies also found that OB showed competitive interaction

and nmol L)1 affinity for the L-type calcium channel.12

The pharmacological profile of OB in our studysuggests that OB might exert its spasmolytic effect

mainly by reducing calcium influx through L-type

calcium channels on human colonic smooth muscle

cells.

After oral administration, OB is not absorbed sys-

temically and pharmacokinetic studies in humans

have found that OB is mainly eliminated by feces

(97.8%) and minimally excreted by urine (0.71%).5,19,20

Animal studies using oral doses similar to those used

in humans show maximal and specific accumulation of

OB in the colonic circular muscle,21 and peak levels in

colonic tissues reaching the micromolar range 8 h after

oral drug administration.22 Accordingly, OB might actat the level of the gastrointestinal tract without

systemic absorption. The pharmacological profile of

the effect of OB in our study shows that OB strongly

inhibits spontaneous RPCs (IC50 = 49.9 nmol L)1 for

OB and 1.00 nmol L)1 for nifedipine) but with lesser

potency than the specific L-type calcium channel

blocker nifedipine (Emax= 84.17% and Emax= 95.35%

respectively). These results suggest similar affinity but

lesser intrinsic activity of OB on L-type channels

compared with nifedipine and agree with our previous

studies on the rat colon.17 The effects of OB and

nifedipine on RPCs in our study are reversed by thespecific L-type Ca2+-channel agonist, BayK8644, con-

firming the involvement of L-type calcium channels in

the effect of OB on RPCs. Stretch is an important

stimulus in the gastrointestinal tract and L-type

calcium channels are gated by voltage, several chem-

ical mediators and stretch.23 We found that stretch

increased the amplitude of spontaneous contractions

and baseline tone in the human sigmoid colon through

non-neural mechanisms, and stretch-induced contrac-

tions and tone were also strongly inhibited by OB.

Inhibitory neurotransmission can be quantified with

the latency observed before the onset of the

off-contraction.13 This latency is caused by stimulationof inhibitory motor neurons releasing NO and a purine

acting on P2Y1 receptors.13 The latency is well-corre-

lated with electrophysiological data where the fast and

the slow component of the IJP is due to a purine acting

on P2Y1 receptors and NO respectively.14 The present

study shows that OB does not modify the latency

suggesting that inhibitory neurotransmission in the

human colon is unaffected by OB. Similar results were

reported in the rat colon where the fast component of

A B

Figure 4 (A) Mechanical recording showing the effect of induced tension on spontaneous motility and tone in control conditions (top trace) and

in the presence of otilonium bromide (OB) (0.1 lmol L)1) (bottom trace). (B) Plot graph showing (top) the amplitude increase of spontaneous

contractions induced by tension and (bottom) the increase of basal tone induced by tension. Data are expressed as mean SEM.




9/12

the IJP was not modified.17 In contrast, we found a

strong inhibitory effect of OB on the amplitude of thetwo main types (on and off) of sigmoid contractions

induced by EFS of excitatory EMNs. The mechanisms

of action of OB on the inhibition of amplitude of EFS

on- and EFS off-contractions both mediated by

stimulation of excitatory EMNs co-releasing ACh and

tachykinins acting on NK2 receptors13 need careful

discussion. Initial in vitro studies in the guinea pig

ileum found OB counteracted the spasmogenic effect of

ACh with an IC50 in the 20100 nmol L)1 range,

similar to that found for atropine in the same exper-

iments.10 This antimuscarinic activity was confirmedin binding studies that show that OB binds with sub

lmol L)1 affinity to many types of muscarinic recep-

tors in different tissues.12 Results from these initial

studies described the pharmacological profile of OB as

a non-competitive antimuscarinic compound.10 Other

in vitro studies demonstrated that OB also behaves as a

potent blocker of neuronal nicotinic ACh receptors

blocking Ca2+ uptake induced by nicotinic agonists

with an IC50in the lmol L)1 range, and suggested that

Figure 5 (A) Plot graph showing the effect of

otilonium bromide (OB) (top) and nifedipine

(bottom) on the amplitude of electrical

stimulation (EFS) induced on-contractions.

(B) Concentrationresponse curves reflecting

the inhibitory effect OB and nifedipine on

the amplitude of the EFS inducedon-contractions at the maximum frequency

of stimulation (40 Hz). Data are expressed

as mean SEM (*P < 0.05 vs previousconcentration).




10/12

blockade of nAChRs at the myenteric plexus might

contribute to the spasmolytic effect of OB.11 In addi-

tion, pharmacological studies on guinea-pig proximal

colon and binding studies on isolated cells transfected

with the human tachykinin NK2receptor demonstrate

that OB acts in the lmol L)1 range as muscarinic and

A B

B

Figure 6 (A) Representative tracings showing electrical stimulation (EFS)off-contractions and (B) EFSon-contractions. Note EFSoffresponses occur

after a period of latency (marked in the tracing). Histograms show the reduction of amplitude of electrical on- and off-contractions by otilonium

bromide and the reversion of this effect by the L-type Ca 2+ channel agonist, BayK8644 (1 lmol L)1). Data are expressed as mean SEM (*P < 0.05,

**P < 0.01, ***P< 0.001).

A B C

KCI

OB 0.01 mol L1

OB 0.01 mol L1

A B C

OB 0.1 mol L1

OB 1 mol L1

OB 10 mol L1

OB 0.1 mol L1

OB 10 mol L1

OB 3 mol L1

OB 10 mol L

1

OB 100 mol L1

7

A

C

B

6

5

4

3

2

1

Fluorescence

F/F

0 5 10 15

Time (s)

75

50

25

08 7

Log (otilonium)

Percentage

ofcontrol

6 5 4

20 25 30

Figure 7 (A) Ca2+ transients induced by KCl. (A) This figure illustrates the change in relative Fluo4 fluorescence in one cell in response to an

application of KCl (75 mmol L)1) (showed with a bar) in the presence of increasing concentrations of otilonium bromide (OB; from 10 nmol L )1 to

100 lmol L)1). (B) Images taken at specific time points and corresponding to the dotted lines in different OB concentrations. (C) Doseresponse data

fitted to a sigmoid curve of relative Fluo4 fluorescence DF/F in response to an application of KCl in the presence of different OB concentrations. Data

are expressed as mean SEM.




11/12

tachykinin NK2receptor antagonist.24 Taken together,

these studies suggest multiple mechanisms of action

for the inhibitory properties of OB to reduce stimulated

motility of intestinal smooth muscle.5,24 In the present

study, we could not distinguish between a putative

effect on muscarinic, nicotinic or tachykinergic recep-tors because the predominant effect of OB in the

human sigmoid colon was probably the blockade of

L-type calcium channels. In agreement with this

hypothesis, nifedipine inhibited both EFS-induced on-

and off-contractions and the L-type Ca2+ channel

activator BayK8644 reversed the inhibitory action of

OB on both EFS on- and off-contractions. Altogether,

our results suggest that the predominant mechanism of

action of OB on these potent contractions induced by

stimulation of excitatory EMNs is exerted through

blockade of L-type calcium channels, the same mech-

anisms that we found for the effect of OB on non-

neural RPCs. L-type calcium channels have different

binding sides to dihydropyridines (nifedipine), ben-

zothiazepines (diltiazem) and phenylalkilamines

(verapamil).25 Previous studies have demonstrated that

OB binds to the diltiazem binding side of the L-type

calcium channel with a minor binding on the dihydro-

pyridine binding side.12 Our results show that BayK, a

dihydropiyidine derivative, is able to counteract the

effects of OB. This is probably due to the interaction

with the fraction of OB bound to the dihydropyridine

binding side or to interactions between the different

binding sides of the channel.25 The stretch sensor of

the L-type calcium channel is located in the alpha(1C)-subunit26 where the binding of these different antag-

onists is located. Contraction of smooth muscle cells

from the gastrointestinal tract depends on extra cellu-

lar calcium influx mainly through L-type calcium

channels and calcium release from intracellular stores.

Entry of calcium through L-type voltage-dependent

Ca2+ channels provides the major source for the

contractile response to ACh in canine circular colonic

smooth muscle.27 In contrast, contraction of human

sigmoid circular smooth muscle cells in response to

NKA requires release of intracellular calcium.28 In our

study, contractions induced by ACh, NKA and SP were

strongly inhibited by OB as were intracellular calcium

transients caused by direct KCl-depolarization of sig-

moid smooth muscle cells.

It is always difficult to establish a correlation

between colonic motility in vivo and in vitro. In vivo

human colonic motility is complex and involves three

main motor patterns to produce the normal mixing

and propulsive motor functions: RPCs, giant migrat-

ing contractions (GMCs) and tone, each with specific

mechanisms of control.29 Spontaneous RPCs are

caused by direct transmission of electrical slow waves

from interstitial cells of cajal to smooth muscle

cells30 and contribute to the mixing of colonic

contents.29 Colonic tone can modulate the mechan-

ical efficacy of RPCs and GMCs.29 In vitro on-con-

tractions correlate well with in vivo simultaneouscontractions, the most common pattern of pressure

activity in the human colon, which can slow transit;

and in vitro off-contractions might be equivalent to

sigmoid GMCs, or high-amplitude propagating con-

tractions causing propulsion of stools and initiation of

defecation.31 Latency of in vitro off-contractions

determines the velocity of propagation of in vivo

GMCs,31 suggesting OB might decrease the amplitude

of sigmoid contractions without affecting their prop-

agation. Previous in vivo studies in humans by

sigmoid manometry found OB reduced sigmoid motil-

ity and enhanced the pain threshold following infla-

tion of a endoluminal balloon,69 agreeing with the

present study. We believe the evaluation of an

antispasmodic drug should include the assessment of

its effects on all these specific motor patterns and its

effects on direct excitability of sigmoid smooth

muscle cells.29 In conclusion, we found a predomi-

nant mechanism of action for the strong spasmolytic

properties of OB on the normal human sigmoid

motility patterns based in its properties as a blocker

of L-type calcium channels mediating calcium influx

on smooth muscle cells. We believe these pharmaco-

logical properties might mediate the clinically proven

effects of OB on the spastic motility disturbancesdescribed in subsets of patients with IBS.

ACKNOWLEDGMENTS

The authors thank Dr Alex Saenz, M. Marti-Gallostra and

J. Cases (Clnica Sagrada Familia) and Dr Xavier Sunol,

Dr Oscar Estrada, Dr Fran Espin, Dr Adolfo Heredia, Dr Eva

Garca, and Dr Lus Antonio Hidalgo (Hospital de Mataro) forproviding human tissue. We also thank Dr S. Evangelista and

Mrs Jane Lewis for revising the manuscript. This study was

supported by a grant from the Fundacio de Gastroenterologia

Dr Francisco Vilardell, the Fundacio Salut del Consorci Santaridel Maresme, the Departament dUniversitats, Recerca i Soci-

etat de la Informacio (2009-SGR-708), the Fondo de Investigac-

iones Sanitarias del Ministerio de Sanidad y Consumo(IF063678-1), the CIDEM (Centre dInnovacio i Desenvolupa-

ment Empresarial) (RDITSIND06-1-0174), BFU2006-05055/BFI

and by Laboratorios Menarini SA. Ciberehd is funded by theInstituto de Salud Carlos III.

CONFLICT OF INTEREST

P. Clave has served as a speaker for Menarini International and

has received research funding from Laboratorios Menarini SA-Menarini Group, Badalona, Spain.




12/12

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Human Sigmoid Colon